Mycoplasma genitalium (MG) contains one of the smallest genomes capable of sustaining self-replication providing a unique opportunity for the study of genes involved in genital tract infection and development of persistent colonization. MG infection has been reported in as many as 25% of urethritis patients and is highly associated with non-gonococcal urethritis in men and pelvic inflammatory disease, cervicitis and tubal factor infertility in women. Recently MG infection has been associated with HIV acquisition and potentially transmission. Interestingly, MG also has been recovered from inflamed joints. Collectively these inflammatory conditions indicate relevant sites to study the MG transcriptome to identify key genes involved in it infectious life cycle. In this context the impact on the host innate immune response can be addressed. At present the majority of the available data on MG has been generated from axenic cultures of lab-adapted MG strains. In this project, we propose to test the overall hypothesis that MG is a causative agent of inflammatory sequelae of the female genital tract and impacts fertility through selective expression of its ~470 putative genes. We hypothesize that the identification of the transcriptome and subsequent proteomic changes associated with initial infection of vaginal epithelial cells (VEC) followed by ascension to the upper tract and dissemination to joints will reveal crucial aspects of the infectious process. Three complimentary aims are proposed to test the hypothesis. 1) A phenotypic and molecular analysis of 24 international low pass contemporary isolates will be performed for comparison to values established for the type strain G37. These studies will analyze both MG and host transcriptomes and then will use proteomics to characterize the MG intracellular survival niche established in novel polarized genital epithelial cell cultures. 2) Causal relationships between MG and pathogenic sequelae will be evaluated in our novel mouse model of vaginal infection with a focus on transcription patterns associated with infection of selected tissues. This approach also will address the potential impact of MG upon fertility and may lead to the development of a small animal model of MG transmission. 3) Using our mouse model we will investigate the impact of MG upon innate immune responses to synthetic TLR agonists and identify host proteins involved in control of vaginal colonization, ascension and dissemination to other tissues. These studies will provide important information about MG physiology in the context of relevant cell types and should identify a set of genes that will serve as targets for intervention and vaccine development. PUBLIC HEALTH RELEVANCE: Mycoplasma genitalium (MG) appears to be the cause of significant reproductive tract disease in men and women but remains understudied. The recent correlation between MG infection and acquisition and potentially transmission of HIV elevates the need to better understand its infectious life cycle and survival niches. Identifying the MG genes involved in host tissue infection and persistence is a first step in this process and will direct development of diagnostics, interventions and vaccines to combat the antibiotic resistant nature of mycoplasmas and will provide important insights into other intracellular bacterial pathogens and basic mechanisms of host immune evasion.